Several multiprotein complexes are implicated in the ATP-dependent remodeling of nucleosomes for transcription, including the evolutionarily conserved S. cerevisiae Snf/Swi complex. Other essential processes also require the temporal restructuring of chromatin as cells progress through the cell division cycle or undergo differentiation, any of which might require Snf/Swi or related factors. A novel sixteen- protein complex capable of remodeling the structure of chromatin (RSC) has been purified from yeast. Despite a shared ATP-dependent nucleosome remodeling activity, the functions of RSC and Snf/Swi are distinct: Rsc polypeptides are indispensable, and thermolabile mutations in two RSC genes, STH1 (SNF2 homolog) and SFH1 (SNF5 homolog), arrest cells at the G2/M transition of the cell cycle. A study of sth1 and sfh1 conditional mutants therefore presents an excellent opportunity to investigate the mechanisms that link chromatin remodeling to progression through the cell division cycle. To identify targets of RSC function, screens for extragenic suppressors of an sth1-ts mutation, including multicopy suppressors and suppressor mutations, and for second-site mutations that are lethal in combination with this ts allele will be completed. In the second aim, new sfh1-ts alleles, identified in a genetic screen and from a targeted mutagenesis based on the corresponding snf5-ts mutations, will be characterized functionally and biochemically. Conditional alleles can lead to partial or complete loss of SFH1 function by impairing distinct activities, including cell cycle progression, G1- specific phosphorylation, transcriptional activation, and assembly into RSC. The premise that the abundance, phosphorylation state, or activities of Sth1p or Sfh1p fluctuate in the cell cycle, will be tested in aim three. sth1 and sfh1 mutants will be assayed for the ability to carry out specific functions implicated in the dynamic reorganization of chromatin during the cell cycle, including those required for the completion of DNA replication or chromosome condensation and segregation. In the fourth objective, mechanism(s) by which RSC restructures nucleosomes will be studied in vivo using a novel probe of the chromatin structure of mini-chromosomes. The long-term goal of this project is to define the physiological function of RCS, including the transduction pathway(s) that links chromatin remodeling to cell cycle progression. Elucidation of this connection will impact directly on our understanding of mechanisms that control differentiation and development in multicellular organisms.